Cyclic guanosine monophosphate (cGMP) plays an essential role in signal transduction to regulate a diverse range of cellular processes, including tumor cell survival. Intracellular cGMP levels are tightly regulated by a family of phosphodiesterase (PDE) isozymes, whereby inhibitors can amplify the magnitude and/or duration of the signal. Previous studies that we have conducted suggest that cGMP PDE inhibition is an important off-target effect, which is responsible for the tumor cell growth inhibitory activity of certain highly potent nonsteroidal anti-inflammatory drugs such as sulindac. These studies led us to hypothesize that safer and more selective cancer drugs can be discovered by interrogating a large compound library for cGMP PDE inhibitory activity. However, it is unclear which PDE isozyme is responsible for the activity of sulindac and few inhibitors are available to identify the specific PDE isozyme(s) involved in carcinogenesis. Towards our long term goal of identifying selective cGMP PDE inhibitors with anti-cancer activity, we propose to develop a HTS assay using a novel cGMP biosensor involving a chimeric protein composed of cyan- and yellow fluorescent proteins (CFP/YFP) fused with the cGMP binding domain of PDE2 or PDE5. Fluorescence resonance energy transfer (FRET) results from a conformational change of the protein upon cGMP binding to cause decreased polarization (anisotropy) of YFP fluorescence, which provides a highly specific analyte to image intracellular cGMP in live cell populations using a laser scanning fluorimeter. As proof of principle, we have shown that known cGMP PDE inhibitors can increase cGMP levels in transiently transfected cells. The specific aims of this proposal are to: 1) generate a stable transfected colon tumor cell line that expresses the cGMP biosensor, 2) develop an image-based assay to measure cGMP levels in transfected cell populations, and 3) configure the assay for HTS and develop secondary assays to assess hit specificity. A key innovation of this cell-based assay is its ability to identify pharmacologically relevant cGMP PDE inhibitors that suppress tumor cell growth without prior knowledge of the specific isozymes involved in carcinogenesis. Colorectal cancer is a major public health problem, although previous studies have shown that certain nonsteroidal anti-inflammatory drugs (NSAIDs) can provide a significant protective benefit. Mechanistic studies suggest that cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition is an important off- target responsible for the growth inhibitory activity of certain highly potent NSAIDs such as sulindac against colon tumor cells. These studies led us to hypothesize that safe and highly selective anticancer drugs can be discovered by interrogating a large compound library for cGMP PDE inhibitory activity. Towards this goal, we propose to develop an innovative cell-based assay involving a fluorescence cGMP biosensor for high throughput screening. [unreadable]